The stable maintenance of a plasmid, particularly at high copy number, is important for the preparation of DNA carrying a therapeutic gene for use in gene therapy. However, extrachromosomal DNA carried in host cells is inherently unstable in cell culture because cultured cells which contain plasmids usually have an increased metabolic burden compared to plasmid-free segregant cells. In efforts to maintain plasmid stability and decrease metabolic burden, plasmids engineered to contain dominant selectable markers have been routinely used. During scale-up fermentation of bacterial or yeast host strains, the presence of the selecting agent prevents plasmid loss and overgrowth by cells not burdened by the effort of replication and maintenance of plasnid DNA.
Antibiotic resistance genes, for example encoding resistance to antibiotics such as ampicillin, kanamycin or tetracycline, are the most common dominant selectable markers used in molecular biology cloning and fermentation procedures for the production of recombinant proteins or plasmid DNA. For continuous fermentation in the presence of an antibiotic, selective pressure is lost because the antibiotic loses activity over time due to culture dilution or degradation by the host cell. Therefore, some of the more successful methods for maintaining plasmids do not utilize antibiotic selection but rather rely on a mutant host which is unable to synthesize an amino acid and inserting the gene which provides for this synthesis in the plasmid. Other solutions which prevent the takeover of a culture by plasmid-free segregant involve placing a gene coding for a toxic product in the chromosome and then including a corresponding repressor system in the plasmid. Plasmid-free cells are effectively killed upon segregation. Even with selective pressure, however, plasmid-free cells may continue to grow due to leakage of the complementing product of the selective gene from plasmid-bearing cells. In addition, the use of genes for antibiotic resistance or other dominant selectable markers on vectors intended for gene therapy has raised potential problems related to expression of those genes in the target mammalian cell or host mammalian organism. Expression of the those genes in the target mammalian cell may lead to its destruction and/or to an antigenic response to the gene product in the mammal. There are also concerns regarding contamination of the final product with the antibiotic used for plasmid selection in culture, with the potential induction of a severe immune response to the antibiotic, e.g., anaphylactic shock. The wide-spread use of bacterial genes for antibiotic resistance also will ultimately result in their transfer to the bacterial population as a whole. There is, therefore, a need for a method of plasmid maintenance that does not require the presence of plasmid borne genes or antibiotic selection.